Dermatitis treatment

ABSTRACT

A compound of formula (I) 
       R-L-CO—X  (I)
 
     (wherein R is a C 10-24  unsaturated hydrocarbon group optionally interrupted by one or more heteroatoms or groups of heteroatoms selected from S, O, N, SO, SO 2 , said hydrocarbon group comprising at least 4 non-conjugated double bonds;
         L is a linking group forming a bridge of 1 to 5 atoms between the R group and the carbonyl CO wherein L comprises at least one heteroatom in the backbone of the linking group; and   X is an electron withdrawing group) or a salt thereof   for use in the treatment of dermatitis.

This invention relates to the use of certain polyunsaturated long-chainketones for the treatment of dermatitis and in particular to ketonescarrying electron withdrawing substituents alpha to the carbonylfunctionality in such treatment. The invention also relates to methodsof treating dermatitis in patients comprising administration of thecompounds of the invention to the patient, ideally topically.

BACKGROUND

In its broadest sense, dermatitis is inflammation of the skin. It is acommon and disfiguring skin condition which requires quick and efficienttreatment. Dermatitis symptoms vary, however, with the different formsof the condition. Symptoms vary from skin rashes to bumpy rashes throughto flaky skin and blisters. Although different types of dermatitis havevarying symptoms, there are certain signs that are common for all ofthem, including redness of the skin, swelling, itching, skin lesions andsometimes oozing and scarring.

Also, the area of the skin on which the symptoms appear tends to bedifferent with every type of dermatitis. Types of dermatitis areclassified according to the cause of the condition. Contact dermatitisis caused by an allergen or an irritating substance. Irritant contactdermatitis accounts for 80% of all cases of contact dermatitis.

Atopic dermatitis is very common worldwide and increasing in prevalence.Atopic dermatitis is a type of eczema and is an inflammatory,chronically relapsing, non-contagious and itchy skin disorder.

Other less common forms of dermatitis include dermatitis herpetiformis.It is characterized by intensely itchy, chronic papulovesiculareruptions, usually distributed symmetrically on extensor surfaces suchas the back of neck, scalp, elbows, knees, back, hairline, groin orface.

Seborrheic dermatitis is a dermatitis that occurs in the vicinity ofsebaceous glands and is caused by sebum over production. The conditiontends to give a scaly, flaky skin condition.

Stasis dermatitis is an inflammation on the lower legs which is causedby build up of blood and fluid and it is more likely to occur in peoplewith varicose veins.

Infective dermatitis is dermatitis secondary to a skin infection. Asummary of dermatitis can be found in Blauvelt et al in Chapter 11 of JAllergy Immunol Feb 2003. Here the author, in particular, discussesallergic and immunological diseases of the skin, such as allergiccontact dermatitis.

The present inventors seek new treatments for all types of dermatitiswith particular emphasis on atopic dermatitis and contact dermatitis.The use of the compounds of the invention in allergic contact dermatitisis particularly considered.

There are numerous therapies for dermatitis on the market. Treatment ofdermatitis is made according to the particular cause of the disease.Creams that contain corticosteroids are frequently used and simplyavoiding the allergens and irritants are part of most treatment plans.For some types of dermatitis, non-steroidal medications may help relievesigns and symptoms. For all types of dermatitis, occasional use ofover-the-counter antihistamines can reduce itching. Calamine lotion typeproducts might be applied to the skin or a barrier cream such as zincoxide or a suntan lotion might be used. For other conditions, a doctormight just advise letting the body's natural mechanisms sort theproblem.

The present inventors have realised that the compounds discussed herein,compounds based upon long chain unsaturated fatty acid molecules, havepotential in the treatment of dermatitis, in particular, atopic orcontact dermatitis.

The compounds proposed for use in this invention have been disclosedbefore, for example, in EP-A-1469859 but only in the treatment ofpsoriasis which is a skin condition but is not a form of dermatitis.Psoriasis has very different biochemistry/immunology. The compounds havealso been suggested, in WO2010/139482, for the treatment ofglomerulonephritis or for the treatment of rheumatoid arthritis inWO2012/028688. The present inventors have realised that these compoundsand others have utility also in the treatment of dermatitis. It isgenerally accepted that the compounds of the present invention induceanti-inflammatory effects through modulation of TNFα initiatedpro-inflammatory intracellular signalling cascades leading totranscription factor NF-κB activation in activated keratinocytes, whichexplains the compounds anti-inflammatory potential in diseases such aspsoriasis and rheumatoid arthritis.

Oxazolone induced contact hypersensitivity in mice is a widely usedmodel for delayed-type hypersensitivity (type IV hypersensitivity) withT-cell driven inflammation following challenge in previously sensitizedskin. Examples of diseases where delayed-type hypersensitivity plays asignificant role for disease development and clinical signs areDermatitis including Contact Dermatitis and Atopic Dermatitis.

Psoriasis is an autoimmune induced, chronic disease of skin,characterized by T-cell accumulation, inflammation andhyperproliferation of keratinocytes in epidermis, as opposed todermatitis being a delayed-type IV hypersensitivity disorder, the latterprimarily driven by MHC class II restricted CD4⁺ T cells.

The present inventors have surprisingly found that after oxazolonechallenge, the compound of the invention surprisingly had markedanti-inflammatory effects shown by the ability to reduce oxazoloneinduced ear swelling. The effect was observed at very low concentrationsof active compound (down to 0.01% active compound in the topicalformulation) and shows that the compounds of the present inventionsurprisingly have anti-inflammatory effects through a previously unknownpathway that significantly inhibit delayed-type hypersensitivitymediated inflammatory pathways.

Consequently, the compounds of the present invention surprisingly haveclinical beneficial effects in dermatitis including Contact Dermatitisand Atopic Dermatitis.

The present inventors have therefore shown that compounds claimed hereinare able to suppress oxazolone induced delayed-type-hypersensitivity(DTH) responses in mice.

SUMMARY OF INVENTION

Thus, viewed from one aspect the invention provides a compound offormula (I)

R-L-CO—X  (I)

(wherein R is a C₁₀₋₂₄ unsaturated hydrocarbon group optionallyinterrupted by one or more heteroatoms or groups of heteroatoms selectedfrom S, O, N, SO, SO₂, said hydrocarbon group comprising at least 4non-conjugated double bonds;

L is a linking group forming a bridge of 1 to 5 atoms between the Rgroup and the carbonyl CO wherein L comprises at least one heteroatom inthe backbone of the linking group; and

X is an electron withdrawing group) or a salt thereof

for use in the treatment of dermatitis.

Viewed from another aspect the invention provides a method of treatingdermatitis comprising administering to an animal, preferably a mammal,in need thereof, e.g. human, an effective amount of a compound offormula (I):

R-L-CO—X  (I)

(wherein R is a C₁₀₋₂₄ unsaturated hydrocarbon group optionallyinterrupted by one or more heteroatoms or groups of heteroatoms selectedfrom S, O, N, SO, SO₂, said hydrocarbon group comprising at least 4non-conjugated double bonds;

L is a linking group forming a bridge of 1 to 5 atoms between the Rgroup and the carbonyl CO wherein L comprises at least one heteroatom inthe backbone of the linking group; and

X is an electron withdrawing group) or a salt thereof.

Viewed from another aspect the invention provides use of a compound offormula (I) or a salt thereof as hereinbefore described for use in themanufacture of a medicament for treating dermatitis.

DETAILED DESCRIPTION

This invention involves the use of compounds of formula (I) or a saltthereof in the treatment of dermatitis and related conditions.

Dermatitis

As noted above, the term dermatitis is broad and covers a variety ofdifferent skin conditions. Whilst it is envisaged that the compounds ofthe invention may have utility in treating a variety of different formsof dermatitis, it is preferred if the compounds are used to treat atopicdermatitis or contact dermatitis.

In particular, the compounds of the invention may be used to treatcontact dermatitis such as allergic contact dermatitis or irritantcontact dermatitis.

The nature of the allergan or irritant which causes the contactdermatitis can vary a lot and many people have different reactions todifferent allergans/irritants.

One of the most common causes of allergic contact dermatitis are plantsof the Toxicodendron genus: poison ivy, poison oak, and poison sumac.Certain alkyl resorcinols such as bilobol found in Gingko biloba fruitsare strong skin irritants. Other allergens include nickel, gold, balsamof Peru (Myroxylon pereirae), and chromium.

Common causes of irritant contact dermatitis are harsh (highly alkaline)soaps, detergents, and cleaning products. Irritant contact dermatitiscan be divided into forms caused by chemical irritants and those causedby physical irritants. Common chemical irritants implicated includesolvents (alcohol, xylene, turpentine, esters, acetone, ketones, andothers); metalworking fluids (neat oils, water-based metalworking fluidswith surfactants); latex; kerosene; ethylene oxide; surfactants intopical medications and cosmetics (sodium lauryl sulfate); alkalies(drain cleaners, strong soap with lye residues). Physical irritantcontact dermatitis may most commonly be caused by low humidity from airconditioning. Also, many plants directly irritate the skin.

A further form of contact dermatitis is photocontact dermatitis. Theskin condition is caused by exposure to ultraviolet light (320-400 nmUVA).

The invention requires the use of a compound of formula (I). The group Rpreferably comprises 5 to 9 double bonds, preferably 5 or 8 doublebonds, e.g. 5 to 7 double bonds such as 5 or 6 double bonds. These bondsshould be non-conjugated. It is also preferred if the double bonds donot conjugate with the carbonyl functionality.

The double bonds present in the group R may be in the cis or transconfiguration however, it is preferred if the majority of the doublebonds present (i.e. at least 50%) are in the cis configuration. Infurther advantageous embodiments all the double bonds in the group R arein the cis configuration or all double bonds are in the cisconfiguration except the double bond nearest the carbonyl group whichmay be in the trans configuration.

The group R may have between 10 and 24 carbon atoms, preferably 12 to 20carbon atoms, especially 17 to 19 carbon atoms.

Whilst the R group can be interrupted by at least one heteroatom orgroup of heteroatoms, this is not preferred and the R group backbonepreferably contains only carbon atoms.

The R group may carry up to three substituents, e.g. selected from halo,C₁₋₆ alkyl e.g. methyl, C₁₋₆ alkoxy. If present, the substituents arepreferably non-polar, and small, e.g. a methyl group. It is preferredhowever, if the R group remains unsubstituted.

The R group is preferably linear. It preferably derives from a naturalsource such as a long chain fatty acid or ester. In particular, the Rgroup may derive from AA, EHA or DHA.

The linking group L provides a bridging group of 1 to 5 backbone atoms,preferably 2 to 4 backbone atoms between the R group and the carbonyl.The atoms in the backbone of the linker may be carbon and/or beheteroatoms such as N, O, S, SO, SO₂. The atoms can form part of a ringand the backbone atoms of the linking group can be substituted with sidechains, e.g. with groups such as C₁₋₆ alkyl, oxo, alkoxy, or halo.

Preferred components of the linking group are —CH₂—, —CH(C₁₋₆alkyl)-,—N(C₁₋₆alkyl)-, —NH—, —S—, —O—, —CH═CH—, —CO—, —SO—, —SO₂— which can becombined with each other in any (chemically meaningful) order to formthe linking group. Thus, by using two methylene groups and an —S— groupthe linker —SCH₂CH₂— is formed. It will be appreciated that at least onecomponent of the linker provides a heteroatom in the backbone.

The linking group L contains at least one heteroatom in the backbone. Itis also preferred if the first backbone atom of the linking groupattached to the R group is a heteroatom or group of heteroatoms.

It is highly preferred if the linking group L contains at least one—CH₂— link in the backbone. Ideally the atoms of the linking groupadjacent the carbonyl are —CH₂—.

It is preferred that the group R or the group L (depending on the sizeof the L group) provides a heteroatom or group of heteroatoms positionedα, β, γ, or β to the carbonyl, preferably β or γ to the carbonyl.Preferably the heteroatom is O, N or S or a sulphur derivative such asSO.

Highly preferred linking groups therefore are —NH₂CH₂, —NH(Me)CH₂—,—SCH₂—, —SOCH₂—, or —COCH₂—

It is also within the invention for the linking group to be a ring or tocomprise a ring. Thus for example, the linker might be thiophene, e.g.2,4-thiophene which provides a two atom bridge to the carbonyl (via theshortest route).

It would also be possible for the linker to comprise a ring such asfuran, tetrahydrofuran, piperidine, cyclohexane, benzene or pyridine.Where the linker comprises a ring it is preferred if this is a 5 or 6membered ring. It is preferred if the ring comprises at least oneheteroatom or group of heteroatoms. It is preferred if the ring isunsaturated or aromatic. When the R and COX groups bind directly to sucha ring, it is preferred if the R group and COX group bind on differentatoms and preferred if they bind on carbon atoms of the ring.

The substitution pattern is preferably such that the R and carbonylsubstituents are alpha, gamma to each other (i.e. 1,3 or 2, 4 or 3,5-split).

For the avoidance of doubt, it is stressed that the 1 to 5 atom bridgeshould be counted as the shortest route from the start of the linker tothe carbonyl.

Suitable ring linkers are shown below where the R group and carbonyl canbind to any two carbon atoms on these rings:

It is also within the scope of the invention for the linker to comprisea ring and non ring portion, e.g. CH₂-thiophene or NH₂-thiophene and soon. In such a linker it is preferred if the R group binds directly tothe ring and that the carbonyl group binds to the non ring portion, e.g.a —CH₂— linkage. The skilled man will be able to devise all kinds ofdifferent linkers suitable for use in the invention.

Highly preferred linking groups are SCH₂, NHCH₂, N(Me)CH₂, 2,4-thiopheneand 2,5-thiophene.

The group X is an electron withdrawing group. Suitable groups in thisregard include O—C₁₋₆ alkyl, CN, OCO₂—C₁₋₆ alkyl, phenyl, CHal₃, CHal₂H,CHalH₂ wherein Hal represents a halogen, e. g. fluorine, chlorine,bromine or iodine, preferably fluorine.

In a preferred embodiment the electron withdrawing group is CHal₃,especially CF₃.

Thus, preferred compounds of formula (I) are those of formula (I′)

R—Y1-Y2-CO—X

wherein R and X are as hereinbefore defined;

Y1 is selected from O, S, NH, N(C₁₋₆-alkyl), SO or SO₂ and

Y2 is (CH₂)_(n) or CH(C₁₋₆ alkyl); or

Y1 and Y2 taken together form a 5 or 6 membered heterocyclic, optionallyunsaturated or aromatic ring; or

Y1 forms a 5 or 6 membered heterocyclic, optionally unsaturated oraromatic ring and Y2 is (CH₂)_(n);

where n is 1 to 3, preferably 1.

Highly preferred compounds for use in the invention are depicted below.

where X is as hereinbefore defined such as CF₃.

The following compounds are highly preferred for use in the invention:

Where possible, the compounds of the invention can be administered insalt, solvate, prodrug or ester form, especially salt form. Preferablyhowever, no such form is used.

Typically, a pharmaceutical acceptable salt may be readily prepared byusing a desired acid. The salt may precipitate from solution and becollected by filtration or may be recovered by evaporation of thesolvent. For example, an aqueous solution of an acid such ashydrochloric acid may be added to an aqueous suspension of a compound offormula (I) and the resulting mixture evaporated to dryness(lyophilised) to obtain the acid addition salt as a solid.Alternatively, a compound of formula (I) may be dissolved in a suitablesolvent, for example an alcohol such as isopropanol, and the acid may beadded in the same solvent or another suitable solvent. The resultingacid addition salt may then be precipitated directly, or by addition ofa less polar solvent such as diisopropyl ether or hexane, and isolatedby filtration.

Suitable addition salts are formed from inorganic or organic acids whichform non-toxic salts and examples are hydrochloride, hydrobromide,hydroiodide, sulphate, bisulphate, nitrate, phosphate, hydrogenphosphate, acetate, trifluoroacetate, maleate, malate, fumarate,lactate, tartrate, citrate, formate, gluconate, succinate, pyruvate,oxalate, oxaloacetate, trifluoroacetate, saccharate, benzoate, alkyl oraryl sulphonates (eg methanesulphonate, ethanesulphonate,benzenesulphonate or p-toluenesulphonate) and isethionate.Representative examples include trifluoroacetate and formate salts, forexample the bis or tris trifluoroacetate salts and the mono or diformatesalts, in particular the tris or bis trifluoroacetate salt and themonoformate salt.

In a further highly preferred embodiment, the compound of the inventionis a sulphonium salt. In such a compound, a sulphur atom in the backboneof the molecule, e.g. in the linker group, is functionalised to carry aC1-6-alkyl group. This can be achieved through reaction with an alkylhalide, e.g. methyl iodide. The halide ion forms the counterion of thesalt.

In a further preferred embodiment therefore the invention provides asulphonium salt of a compound of formula (I). Preferably the compound isof formula (VI)

RS(C₁₋₆alkyl)CH₂—COX⁺Z⁻  (VI)

where R and X are as hereinbefore defined and Z is a counterion, e.g.halide; e.g. the compound

Compounds of formula (I) may be manufactured using known chemicalsynthetic routes. It is convenient to begin synthesis from thecommercially available compounds arachidonic acid (AA), EPA(all-Z-eicosa-5,8,11,14,17-pentaenoic acid) or DHA(all-Z-docosa-4,7,10,13,16,19-hexaenoic acid). Conversion of the acidfunctionality of these compounds into, for example a —COCF₃ group can beachieved readily, e.g. by converting the carboxylic acid into itscorresponding acid chloride and reacting the same with trifluoroaceticanhydride in the presence of pyridine.

Introduction of a heteroatom into the carbon chain is also achievedreadily. Conveniently, for example, the starting acid is reduced to analcohol and, if required, converted to the corresponding thiol. Thenucleophilic thiol may then be reacted with a group such as BrCH₂COCF₃thereby introducing the carbonyl and electron withdrawing species.Complete synthetic protocols may be found in J. Chem. Soc., Perkin Trans1, 2000, 2271-2276 or J. Immunol., 1998, 161, 3421.

Where the backbone of the molecule contains a nitrogen atom, analternative synthesis is required. Formation of a polyunsaturatedalcohol can be achieved using protocols give in the above Perkin Transpaper. Thereafter, conversion of an alcohol —OH to —NH₂ with, forexample, phthalimide and subsequent hydrazine reduction allows formationof a —NH₂CH₂COCF₃ group by reaction with trifluoropropyleneoxide (TFPO)and oxidation of the hydroxyl to a ketone. This reaction is shown below.

Methylation of the nitrogen can be effected before this reaction by theformation of an N—BOC group and reduction, e.g. with lithium aluminiumhydride. Reaction with TFPO and oxidation yields the linker NMe-CH₂.

The compounds of the invention are proposed primarily for use in thetreatment of, inter alia, dermatitis.

By treating or treatment is meant at least one of:

(i). preventing or delaying the appearance of clinical symptoms of thedisease developing in a mammal;(ii). inhibiting the disease i.e. arresting, reducing or delaying thedevelopment of the disease or a relapse thereof or at least one clinicalor subclinical symptom thereof, or(iii). relieving or attenuating one or more of the clinical orsubclinical symptoms of the disease.

The benefit to a subject to be treated is either statisticallysignificant or at least perceptible to the patient or to the physician.In general a skilled man can appreciate when “treatment” occurs. It isparticularly preferred if the compounds of the invention are usedtherapeutically, i.e. to treat a condition which has manifested ratherthan prophylactically. It may be that the compounds of the invention aremore effective when used therapeutically than prophylactically.

The compounds of the invention can be used on any animal subject, inparticular a mammal and more particularly to a human or an animalserving as a model for a disease (e.g., mouse, monkey, etc.).

In order to treat a disease an effective amount of the active agentneeds to be administered to a patient. A “therapeutically effectiveamount” means the amount of a compound that, when administered to ananimal for treating a state, disorder or condition, is sufficient toeffect such treatment. The “therapeutically effective amount” will varydepending on the compound, the disease and its severity and the age,weight, physical condition and responsiveness of the subject to betreated and will be ultimately at the discretion of the attendantdoctor.

It may be that to treat dermatitis according to the invention that thecompound of formula (I) has to be reapplied at certain intervals.Suitable dosage regimes can be prescribed by a physician.

While it is possible that, for use in the methods of the invention, acompound of formula I may be administered as the bulk substance, it ispreferable to present the active ingredient in a pharmaceuticalformulation, for example, wherein the agent is in admixture with apharmaceutically acceptable carrier selected with regard to the intendedroute of administration and standard pharmaceutical practice.

The term “carrier” refers to a diluent, excipient, and/or vehicle withwhich an active compound is administered. The pharmaceuticalcompositions of the invention may contain combinations of more than onecarrier. Such pharmaceutical carriers are well known in the art. Thepharmaceutical compositions may also comprise any suitable binder(s),lubricant(s), suspending agent(s), coating agent(s), and/or solubilizingagent(s) and so on. The compositions can also contain other activecomponents, e.g. other drugs for the treatment of dermatitis.

The active agent of the invention may therefore be combined withsteroids or barrier materials (such as zinc oxide).

It will be appreciated that pharmaceutical compositions for use inaccordance with the present invention may be in the form of oral,parenteral, transdermal, sublingual, topical, implant, nasal, orenterally administered (or other mucosally administered) suspensions,capsules or tablets, which may be formulated in conventional mannerusing one or more pharmaceutically acceptable carriers or excipients.The compositions of the invention could also be formulated asnanoparticle formulations.

However, for the treatment of dermatitis, the compositions of theinvention will preferably be applied topically. They may be in the formof ointments, gels, syrups, creams and so on. Any conventional topicalformulation vehicle may be used.

The pharmaceutical compositions of the invention may contain from 0.01to 99% weight—per volume of the active material.

A therapeutically effective amount of the compound of the presentinvention can be determined by methods known in the art. Thetherapeutically effective quantities will depend on the age and on thegeneral physiological condition of the patient, the route ofadministration and the pharmaceutical formulation used. The therapeuticdoses will generally be between about 10 and 2000 mg/day and preferablybetween about 30 and 1500 mg/day. Other ranges may be used, including,for example, 50-500 mg/day, 50-300 mg/day, 100-200 mg/day.

Administration may be once a day, twice a day, or more often, and may bedecreased during a maintenance phase of the disease or disorder, e.g.once every second or third day instead of every day or twice a day. Thedose and the administration frequency will depend on the clinical signs,which confirm maintenance of the remission phase, with the reduction orabsence of at least one or more preferably more than one clinical signsof the acute phase known to the person skilled in the art.

The compounds of the invention may be used to treat dermatitis incombination with other known pharmaceuticals for said purpose and thisforms a further aspect of the invention. Other useful pharmaceuticalsinclude corticosteroids and anti-histamines.

The invention is described further below with reference to the followingnon-limiting examples and figures.

DESCRIPTION OF FIGURES

FIG. 1 shows the absolute increase in ear thickness of the right ear vs.the left ear for the oxazolone-induced ear edema model in mice atdifferent therapeutic concentrations of AVX001.

FIG. 2 shows the relative increase in ear thickness of the right ear vsthe left ear for the oxazolone-induced ear edema model in mice atdifferent therapeutic concentrations of AVX001.

FIG. 3 shows the significant reduction (20%) in ear tissue PGE2concentration in AVX-treated versus placebo ear tissue.

EXAMPLES

The following compounds were used in the Experiments:

Example 1 AVX001 in the Suppression of Oxazolone-InducedDelayed-Type-Hypersensitivity (DTH) Responses

DTH can be induced by skin sensitization with oxazolone, followed by atopical ear challenge with oxazolone 7 days later. This challengeresults in development of an inflammatory response in the ear within 24hours, which is dependent on antigen-specific T cells. The DTH reactionis associated with the recruitment of a variety of inflammatory cellsand development of edema. The latter can be measured by quantifyingchanges in ear thickness before and after ear challenge.

The oxazolone-induced ear edema model in mice is therefore a model ofdelayed contact hypersensitivity that permits the quantitativeevaluation of the topical and systemic anti-inflammatory activity of acompound following topical administration.

Experimental

Female mice (6-8 wks—BALB/c) were acclimatization for 7 days in 12 hourslight/12 hours dark cycle, at Temperature: 20-24° C., humidity: 55%±10%,on a standard laboratory mouse diet.

Delayed-Type-Hypersensitivity

Day 0: Mice were anesthetized by inhalation of 3-4% isoflurane in a 1:1mixture of oxygen and medical air. The abdominal skin was shaved toenable topical application of 100 μl 1.5% (w/v) oxazolone in 100%acetone.

Day 7: All ears were wiped with ethanol to optimize exposure tooxazolone. Subsequently, the right ear of the sensitized animals werechallenged by topical administration of 20 μl (10 μl on each side of theear) 2.5% (w/v) oxazolone in 100% ethanol. The left ears were treatedwith 20 μl 100% ethanol.

Day 8: After 24 hours, mice were sacrificed and the right ears werecollected to enable analysis by (immuno)-histochemistry and/orhistology.

The vehicle used to carry AVX001 was paraffin. The placebo was paraffin.Approx 20 μl (10 μl on each side of the right ear) of composition isapplied to effect the test.

Therapeutic Testing

4 groups (8 mice per group) were studied.

Group 1: Placebo, 1 hour and 7 hours after challenge

Group 2: Compound 0.2 wt %, 1 hour and 7 hours after challenge

Group 3: Compound 0.05 wt %, 1 hour and 7 hours after challenge

Group 4: Compound 0.01 wt %, 1 hour and 7 hours after challenge

Measurements:

Day 7: Ear thickness of both ears was measured directly before theear-challenge (5 measurements per ear) using a Mitutoyo micrometer(accuracy 0.01 mm)

Day 8: 24 Hours after challenge, thickness of left and right ears wasmeasured again.

The increase in ear thickness of each ear was calculated by subtractingthe mean ear thickness measured on day 7 (before challenge) from thethickness on day 8 (24 hrs after challenge). To determine the oxazolonespecific increase in ear thickness, the calculated increase in earthickness of the left ear was subtracted from that of the right ear.Both absolute (Delta μm) and relative (%) increases in ear thicknesswere calculated.

Increase in Ear Thickness (Delta μm):

Thickness day 8 (μm)−Thickness day 7 (μm)

Oxazolone Specific Increase (Delta μm):

Increase right ear (Delta μm)−Increase left ear (Delta μm)

Relative Increase in Ear Thickness (%):

(Increase in ear thickness (Delta μm)/Thickness day 7 (μm))*100%

Relative Oxazolone Specific Increase in Ear Thickness (%):

Relative increase right ear (%)−Relative increase left ear (%)

Results: In the placebo group a significant (p<0.0001) increase of 95%in ear thickness was observed, of the oxazolone challenged ear, comparedwith the ethanol challenged ear. The average thickness of the right earsincreased from 230 μm on day 7 to 447 μm on day 8.

Effect of Treatment with AVX001

Treatment with AVX001 reduced the oxazolone specific ear swelling from217 μm (placebo) to 134 μm (AVX001 0.01%), 103 μm (AVX001 0.05%) and 94μm (AVX001 0.2%). The reduction of the oxazolone specific ear swellingcaused by AVX001 was statistically significant for all concentrations.

Results are presented graphically in FIG. 1+2. Data is also presented intable 1 and 2.

Table 1/2 Summarizing Tables

TABLE 1 Absolute oxazolone specific ear swelling (Δ μm) Absolute AVX001AVX001 AVX001 Increase Placebo 0.01% 0.05% 0.2% Average 217 134 103 94SD 64 43 54 50 n 8 8 8 8 SEM 23 15 19 18

TABLE 2 Relative oxazolone specific ear swelling % Absolute AVX001AVX001 AVX001 Increase Placebo 0.01% 0.05% 0.2% Average 95 58 43 42 SD30 19 23 24 N 8 8 8 8 SEM 11 7 8 9

Example 2 Reduction in Ear Tissue PGE2 Enzyme after Treatment withAVX001

FIG. 3 shows the significant reduction (20%) in ear tissue PGE2concentration in AVX-treated versus placebo ear tissue. PGE2 is ametabolite of arachidonic acid and the results show that the inhibitorAVX001 has hit its target, the group IVa PLA2 enzyme.

Materials and Methods

(i) PGE2 EIA Analysis

Using a PreCellys homogenisator, the right ear biopsies were homogenizedin 400 μl lysisbuffer containing 10 μM indomethasin. The lysate wasstored at −80° C. until further processing. PGE2 EIA analysis earhomogenates was performed according to kit protocol (Cayman Chemicals).Plasma samples were diluted 1:60 and 1:180 in EIA buffer. The sampleswere hybridized over-night (18 hrs, 4° C.). The EIA plates were readusing a Multiscan plate reader (Ascent Labsystems, OD550 nm). Ascentsoftware for Multiscan, Version 2.4.1 was used to obtain the data. PGE2results are shown as mean±SD relative to the paraffin-treated placebomice (n=5 mice).

(ii) Statistical Analysis

Data of groups was examined by one-way analysis of variance, andindividual groups were then compared with Dunnets test or Studentst-test. Data was given as mean±SD, if not stated otherwise. Values ofp<0.05 were considered significant.

1-18. (canceled)
 19. A method of treating dermatitis comprisingadministering to an animal in need thereof an effective amount of acompound of formula (I):R-L-CO—X  (I) wherein R is a C₁₀₋₂₄ unsaturated hydrocarbon groupoptionally interrupted by one or more heteroatoms or groups ofheteroatoms selected from S, O, N, SO, SO₂, said hydrocarbon groupcomprising at least 4 non-conjugated double bonds; L is a linking groupforming a bridge of 1 to 5 atoms between the R group and the carbonyl COwherein L comprises at least one heteroatom in the backbone of thelinking group; and X is an electron withdrawing group, or a saltthereof.
 20. A method of claim 19 wherein the hydrocarbon group R has 5to 7 double bonds.
 21. A method of claim 19 wherein in the hydrocarbongroup R no double bond is conjugated with the carbonyl group.
 22. Amethod of claim 19 wherein in the hydrocarbon group R all double bondsare in the cis configuration or wherein in the hydrocarbon group alldouble bonds are in the cis configuration except the double bond nearestthe carbonyl.
 23. A method of claim 19 wherein the R group comprises 17to 19 carbon atoms.
 24. A method of claim 19 claim wherein the linkinggroup L comprises —CH₂—, —CH(C₁₋₆alkyl)-, —N(C₁₋₆alkyl)-, —NH—, —S—,—O—, —CH═CH—, —CO—, —SO—, or —SO₂— which can be combined with each otherin any chemically meaningful order to form the linking group.
 25. Amethod of claim 19 wherein L comprises at least one heteroatom or groupof heteroatoms in the backbone of the linking group selected from O, S,N, or SO.
 26. A method of claim 19 wherein L is —NH₂CH₂, —NH(Me)CH₂—,—SCH₂—, —SOCH₂—, 2,4-thiophene and 2,5-thiophene.
 27. A method of claim19 wherein X is O—C₁₋₆ alkyl, CN, OCO₂—C₁₋₆ alkyl, phenyl, CHal₃,CHal₂H, CHalH₂ wherein Hal represents a halogen.
 28. A method of claim19 wherein X is CHal₃, CHal₂H or CHalH₂ wherein Hal represents fluorine.29. A method of claim 19 wherein X is CHal₃.
 30. A method of claim 19wherein X is CF₃.
 31. A method of claim 19 wherein the compound offormula (I) has the formula:

wherein X is X is an electron withdrawing group.
 32. A method of claim31 wherein X is CF₃.
 33. A method of claim 19 wherein the compound hasthe formula (I′)R—Y1-Y2-CO—X  (F) wherein in formula (I′): R is a C₁₀₋₂₄ unsaturatedhydrocarbon group optionally interrupted by one or more heteroatoms orgroups of heteroatoms selected from S, O, N, SO, SO₂, said hydrocarbongroup comprising at least 4 non-conjugated double bonds; X is anelectron withdrawing group; Y1 is selected from O, S, NH, N(C₁₋₆-alkyl),SO or SO₂ and Y2 is (CH₂)_(n) or CH(C₁₋₆ alkyl); or Y1 and Y2 takentogether form a 5 or 6 membered heterocyclic, optionally unsaturated oraromatic ring; or Y1 forms a 5 or 6 membered heterocyclic, optionallyunsaturated or aromatic ring and Y2 is (CH₂)_(n); where n is 1 to
 3. 34.A method of claim 33 wherein n is
 1. 35. A method of claim 19 whereinthe compound of formula (I) is AVX001.
 36. A method of claim 19 whereinthe dermatitis is contact dermatitis or atopic dermatitis.
 37. A methodof claim 19 wherein the dermatitis is allergan contact dermatitis.
 38. Amethod of claim 19 wherein the compound is applied topically.
 39. Amethod of claim 19 wherein the animal is a mammal.
 40. A method of claim19 wherein the animal is a human.
 41. A method of treating dermatitiscomprising administering to a mammal in need thereof an effective amountof AVX001.
 42. A method of claim 41 wherein the dermatitis is contactdermatitis or atopic dermatitis.
 43. A method of claim 41 wherein thedermatitis is allergan contact dermatitis.
 44. A method of claim 41wherein the mammal is a human.